Regulation of Clathrin and Adaptor Function

描述(由申请人提供)：膜定义了真核细胞与其环境之间以及细胞本身之间的明确的结构和功能边界。物质和信息在这些隔室之间的移动在很大程度上是通过囊泡运输来调节的，而蛋白质涂层总是与这些过程有关，其中笼状蛋白涂层是一种范例。肌醇磷脂被认为是膜转运的重要调节者，识别肌苷在作用部位局部聚集或产生的机制，以及它们如何与效应蛋白相互作用是一个悬而未决的问题。在上一次获奖期间，我们发现了一个II类PI 3-激酶，PI3K-C2？，它能与笼状蛋白特异结合。在过度表达时，PI3K-C2？可以诱导大量细胞内含有这两种蛋白质的笼状蛋白涂层的芽增殖，这些芽移动得非常快。我们最近的工作表明，在没有外源PI3K-C2？的情况下，也可以在活细胞中检测到类似的快速移动结构。表达结果表明，它们含有笼蛋白、PI3K-C2？和胞内转铁蛋白，以及与膜循环有关的相关蛋白。这项建议通过以下方式来检验这一假说：这些“快速网状蛋白”(F-cathrin)结构参与膜循环和内吞途径中的其他步骤：(1)检验F-cathrin结构是具有独特接头、货物和马达蛋白的涂层膜管的假说，并在超微结构水平上探讨它们与外周分选内小体的关系；(2)使用快速成像和同时获取多个信号以及可光激活的GFP探针来确定货物如何通过活细胞中的F-cathrin隔室；(3)解剖单个F-蛋白组分和小G蛋白及其激活剂和效应物对完整细胞中F-蛋白的形成和货物负荷的调节；以及通过结晶学和溶液研究的笼状蛋白重链末端结构域、连接体和脚踝，促进这两种蛋白质的显性-负性形式的产生。虽然哺乳动物细胞膜运输的途径被广泛了解，但它们在某些步骤中的确切功能在机械上，甚至在形态上仍然是未知的，将通过这项工作来解决。与公共卫生的相关性：该项目侧重于单个细胞维持其复杂的细胞内结构并在专门隔间中执行功能的贩运途径。这些途径在所有真核细胞中普遍存在，因此它们是细胞生物学中的中心问题，与许多正常的生物学过程有关，如信号传递、发育和组织中的细胞间通讯。相应地，它们也是许多疾病过程中错乱的部位，包括癌症、心血管疾病和神经疾病。

DESCRIPTION (provided by applicant): Membranes define distinct structural and functional boundaries between the eukaryotic cell and its environment, and within the cell itself. Movement of material and information between these compartments is largely mediated by vesicular transport, and protein coats, of which the clathrin coated membrane is a paradigm, are invariably associated with these processes. Phosphoinositides are recognized to play important roles as regulators of membrane trafficking, and identifying mechanisms by which inositides are locally accumulated or generated at sites of action, and how they interact with effector proteins are unresolved questions. During the last award period we found that a Class II PI 3-kinase, PI3K-C2?, binds specifically to clathrin. Upon overexpression, PI3K-C2? can induce the proliferation of numerous intracellular clathrin coated buds containing both proteins and which move extremely rapidly. Our more recent work has demonstrated that similar rapidly moving structures can be detected in live cells in the absence of exogenous PI3K-C2? expression, that they contain clathrin, PI3K- C2?, and endocytosed transferrin, as well as associated proteins suggesting involvement in membrane recycling. This proposal tests the hypothesis that these `Fast-clathrin' (F-clathrin) structures are involved in membrane recycling and other steps in the endocytic pathway by (1) testing the hypothesis that F- clathrin structures are coated membrane tubules with unique adaptors, cargos and motor proteins, and probing their relationship to peripheral sorting endosomes at the ultrastructural level; (2) using rapid imaging and simultaneous acquisition of multiple signals as well as photoactivatable GFP probes to determine how cargo passes through the F-clathrin compartment in live cells; (3) dissecting the regulation of F-clathrin formation and cargo loading in intact cells by individual F-clathrin components and by small G proteins and their activators and effectors; and (4) elucidating the interactions between PI3K-C2? and the clathrin heavy chain terminal domain, linker and ankle by crystallography and solution studies, facilitating the production of dominant-negative forms of both proteins. Although the pathways of membrane trafficking in mammalian cells are broadly understood, exactly how they function at some steps mechanistically, and even morphologically, remain obscure and will be addressed by this work. Public Health Relevance: This project focuses upon the trafficking pathways by which individual cells maintain their complex intracellular structure and carry out functions in specialized compartments. These pathways are ubiquitous in all eukaryotic cells, and thus these are central issues in cell biology with relevance for many normal biological processes such as signaling, development and intercellular communication in tissues. Correspondingly, they also are sites of derangement in many disease processes including cancer, cardiovascular and neurological diseases.